Spontaneous, rhythmic subsarcolemmal local Ca2+ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca2+ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2+ and cAMP/PKA regulatory components of automaticity are coupled via Ca2+ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca2+-activated ACisoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca2+ over the entire physiological Ca2+ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca2+ cycling proteins drive sarcoplasmic reticulum Ca2+ releases, which, in turn, activate ACs. This feed forward fail safe system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells. Recent studies indicate that basal phosphodiesterase (PDE) activity is increased in sinoatrial nodal cells (SANC) compared to ventricular myocytes (VM), and that the former express, Ca2+-activated adenylyl cyclases (AC), in addition to Ca-inhibited Type V AC. In SANC Ca2+ activates ACs and Ca-activated AC activity is present both in membrane microenvironments that are both rich in caveolin and in those with relatively low caveolin. Localized PDE activity can serve as a functional barrier to limit the diffusion of distal cAMP from the source of its generation by AC. The basal membrane microenvironment of AC activity may indicate that differing AC:PC activities occur within different membrane microenvironments and across the broad range of AC activity microenvironment, within SANC. Furthermore, with respect to G-protein-coupled receptor stimulation, beta-AR and ACs G proteins co-segregate with the high caveolin microdomain, i.e. within caveoli, the AC:PDE activity might be expected to be higher in caveolin-rich microenvironments than in areas in which caveolin is expressed to a lesser extent within both cell types: The purpose of the present study is to determine the distribution of PDE activity in both SANC and VM in lipid raft domains separated by sucrose gradient density configuration, and the AC:PDE within these lipid microdomains.